Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts. In particular, it relates to a genetic marker that is useful for diagnosing or treating spondyloepimetaphyseal dysplasia and for identifying genetic carriers of heritable alleles associated with spondyloepimetaphyseal dysplasia.
2. Discussion of the Related Art
Osteochondrodysplasias are a genetically heterogeneous group of disorders related to cartilage producing cells. Abnormalities in cartilage formation can cause defects in bone deposition, skeletal development, linear growth, and the continued maintenance of cartilage and bone. (Reviewed in Mundlos, S. and Olsen, B. R., Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis, FASEB J. 11(4):227-33 [1997]).
There are numerous and disparate causes of osteochondrodysplasias. (Horton, W. A., Molecular genetic basis of the human chondrodysplasias, Endocrinol. Metab. Clin. North Am. 25(3):683-97 [1996]). A significant number are due to defects in the collagen genes themselves. (Reviewed in Williams, C. J. and Jiminez, S. A., Heritable diseases of cartilage caused by mutations in collagen genes, J. Rheumatol. Suppl. 43:28-33 [1995]; Byers, P. H., Molecular genetics of chondrodysplasias, including clues to development, structure, and function, Curr. Opin. Rheumatol. 6(3):345-50 [1994]). In addition to collagen defects, several forms of osteochondrodysplasias are caused by mutations in the cartilage oligomeric matrix protein (COMP). (Ikegawa, S., et al., Novel and recurrent COMP mutations in preudoachondroplasia and epiphyseal dysplasia, Hum. Genet. 103(6):633-8 [1998]; Briggs, M. D., et al., Diverse mutations in the gene for cartilage oligomeric matrix protein in the pseudoachondroplasia-multiple epiphyseal dysplasia disease spectrum, Am. J. Hum. Genet. 62(2):311-9 [1998]; Briggs, M. D., et al., Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage matrix protein gene, Nat. Genet. 10(3):330-6 [1995]; Ballo, R., et al., Multiple epiphyseal dysplasia, ribbing type: a novel point mutation in the COMP gene in a South African family, Am. J. Med. Genet. 68(4):396-400 [1997]).
Still other osteochondrodysplasias have been found to be caused by defects in secreted peptide growth factors and their receptors. (E.g., Thomas, J. T., et al., A human chondroplysplasia due to a mutation in a TGF-xcex2 superfamily member, Nat. Genet. 12(3):315-7 [1996]; Bonaventure, J., et al., Common mutations in the gene encoding FGFR-3 account for achondroplasia, hypochondroplasia and thanatophoric dysplasia, Acta Paediatr. Suppl. 417:33-38 [1996]).
Finally, mutations in genes which affect protein sulfation cause some forms of osteochondrodysplasia. Protein sulfation is a post-translational modification carried out by all cells. (Lipmann, F., Biological sulfate activation and transfer, Science 128:575-580 [1958]). The primary source of sulfur for the sulfation pathway is free sulfate, which can be transported into the cytoplasm by one of a variety of transmembrane symporter or antiporter molecules. (Elgavish, A., et al., Sulfate transport in human lung fibroblasts (IMR-90), J. Cell Physiol. 125:243-250 [1985]; Markovich, D., et al., Expression cloning of rat renal Na+/SO4(2xe2x88x92) cotransport, Proc. Natl. Acad. Sci. U.S.A. 90:8073-8077 [1993]; Bissig, M., et al., Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes, J. Biol.Chem. 269:3017-3021 [1994]; Hastbacka, J., et al., The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping, Cell 78:1073-1087 [1994]; Everett, L. A., et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17:411-422 [1997]).
Within the cytoplasm, sulfate is activated to a high energy form in two enzymatic steps (Geller, D. H., et al., Co-purification and characterization of ATP-sulfurylase and adenosine-5xe2x80x2-phosphosulfate kinase from rat chondrosarcoma, J. Biol. Chem. 262:7374-7382 [1987]). First, utilizing ATP and sulfate as substrates, an ATP sulfurylase activity catalyzes the synthesis of adenosine 5xe2x80x2-phosphosulfate (APS). Subsequently, an APS kinase activity catalyzes the phosphorylation of the APS to generate 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate (PAPS). PAPS is the universal bioactivated sulfate donor used in all known post-translational sulfation reactions.
Secreted extracellular matrix proteins are post-translationally sulfated in the Golgi, and delivery of PAPS to the Golgi is mediated by a PAPS translocase activity. Microsomal proteins with PAPS binding activity have been identified (Mandon, E. C., et al., Purification of the golgi adenosine 3xe2x80x2-phosphate 5xe2x80x2-phosphosulfate transporter, homodimer within the membrane, Proc. Natl. Acad. Sci. U.S.A. 91:10707-10711 [1994]; Ozeran, J. D., et al., Kinetics of PAPS translocase: evidence for an antiport mechanism, Biochemistry 35:3685-3694 [1996]), but the tissue specificity and the contribution of these proteins to PAPS transport remains unknown. Following transport, sulfation reactions are carried out by substrate-specific sulfotransferases. A major class of sulfation substrates within the Golgi is the side-chains of proteoglycans, which are abundant structural proteins of the extracellular matrices of many tissues. Proteoglycans are particularly abundant in the extracellular matrix of cartilage.
Direct evidence that sulfation of extracellular matrix proteins is essential for proper matrix function was revealed by the identification of mutations in the diastrophic dysplasia sulfate transporter, DTDST. (Hastbacka et al. [1994]). Mutations in the DTDST gene produce a spectrum of recessively inherited osteochondrodysplasia phenotypes. (Hastbacka et al. [1994]; Hastbacka, J., et al., Atelosteogenesis type II is caused by mutations in the diastrophic dyplasia sulfate transporter gene (DTDST): Evidence for a phenotypic series involving three chondrodysplasias, Am. J. Hum. Genet. 58:255-262 [1996]; Superti-Furga, et al., A family of chondrodysplasias caused by mutations in the diastrophic dysplasia transporter gene and associated with impaired sulfation of proteoglycans, Ann. N.Y. Acad. Sci. 785:195-201 [1996]). The severity of the three known disorders, i.e., the moderately severe diastrophic dysplasia phenotype and the lethal forms, atelosteogenesis type II and achondrogenesis type IB, is correlated with the consequences of the mutations on the activity of the transporter. (Superti-Furga, A, et al. Achondrogenesis type IB is caused by mutations in the diastrophic dysplasia sulfate transporter gene, Nature Genet. 12:100-02 (1996). The mutations lead to dramatically reduced proteoglycan sulfation in cartilage, particularly the chondroitin sulfate side chains of aggrecan. However, even in the most severe disorder in the group, some proteoglycan sulfation can be measured. (Rossi A., et al., Undersulfation of proteoglycans synthesized by chondrocytes from patient with achondrogenesis type 1B homozygous for an L483P substitution in the diastrophic dysplasia sulfate transporter, J. Biol. Chem. 271:18456-64 [1996]; Rossi, A., et al., Undersulfation of cartilage proteoglycans ex vivo and increased contribution of amino acid sulfur to sulfation in vitro in McAlister dysplasia/atelosteogenesis type 2, Eur. J. Biochem. 248:741-47 [1997]; Rossi, A., et al., Proteoglycan sulfation in cartilage and cell cultures from patients with sulfate transporter chondrodyplasias: relationship to clinical severity and indications on the role of cellular sulfate production, Matrix Biology 17:361-69[1998]).
Biochemical evidence that a defect in another step in the sulfation pathway can produce an osteochondrodysplasia phenotype was provided by studies in the brachymorphic (bm) mouse. The brachymorphic phenotype is characterized by disproportionate short-limb dwarfism, a short spine and tail, and a domed skull. (Lane and Dickie [1968]). The brachymorphic mouse also exhibits an increased bleeding time, but tests of platelet function, including aggregation and secretion, have so far failed to reveal specific functional deficits in brachymorphic platelets. (Rusiniak, M. E., et al., Molecular markers near the mouse brachymorphic (bm) gene, which affects connective tissues and bleeding time, Mamm. Genome 7:98-102[1996]).
In brachymorphic mice, abnormal growth plates with a structurally abnormal cartilage extracellular matrix and short chondrocyte columns, with comparatively unaligned cells, are apparent on histologic analysis. (Lane and Dickie [1968]; Orkin et al. [1976]; Orkin et al. [1977]; Miller, W. A. and Flynn-Miller, K. L., Achondroplastic, brachymorphic and stubby chondrodystophies in mice, J. Comp. Pathol. 86:349-63 [1976]). Cartilage from this recessively inherited mutant phenotype shows small, diffuse proteoglycan granules and reduced staining for sulfated glycosaminoglycans, consistent with a defect affecting sulfation of the proteoglycans of the cartilage extracellular matrix. (Lane, P. and Dickie, M. M., Three recessive mutations producing diproportionate dwarfing in mice: achondroplasia, brachymorphic, and stubby, J. Hered. 59:300-08 [1968]). In brachymorphic mice, proteoglycan granules show a 50% reduction in size in the reserve zone of the growth plate matrix, and are difficult to identify in the proliferative and hypertrophic zones. (Orkin, R. W., et al., Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice, Dev. Biol. 50:82-94 [19761; Orkin, R. W., et al., Defects in the cartilaginous growth plates of brachymorphic mice, J. Cell Biol. 73:287-99 [1977]). This suggests that reduced proteoglycan sulfation affects the signals that regulate growth plate chondrocyte maturation. Heparan sulfate proteoglycans have been implicated in the sequestration and presentation of growth factors, particularly fibroblast growth factors, to receptors at the cell surface. (Rapraeger, A. C., et al., Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation, Science 252:1705-08 [1991]; Yayon, A., et al., Cell surface, heparin-like molecules are required for binding of basic Fibroblast Growth Factor to its high-affinity receptor, Cell 64:841-48 [1991]; Schlessinger, J., et al., Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors?, Cell 83:357-60 [1995]). Skeletal defects in mice lacking heparan sulfate 2-sulfotransferase include dwarfism, with shortened long bones, ribs and spine, implying a specific role for heparan sulfate proteoglycans in skeletal development. (Bullock, S. I., et al., Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase, Genes Dev. 12:1894-1906 [1998]).
Indeed, reduced activities of both ATP sulfurylase and APS kinase, and decreased synthesis of chondroitin sulfate, have been demonstrated in bm mice. (Schwartz, N. B., et al., Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice, Biochem. Biophys. Res. Commun. 82:173-78 [1978]; Sugahara, K. and Schwartz, N. B., Defect in 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate formation in brachymorphic mice. Proc. Natl. Acad. Sci. U.S.A. 76:6615-18 [1979]). The ATP sulfurylase and APS kinase activities in brachymorphic mouse tissues co-purified. (Geller, D. H., et al. [1987]; Lyle, S., et al., Rat chondrosarcoma ATP sulfurylase and adenosine 5xe2x80x2-phosphosulate kinase reside on a single bifunctional protein, Biochemistry 33:5920-25 [1994]). The functional activity of the APS kinase was reduced to a greater extent than was that of the ATP sulfurylase, suggesting that brachymorphism resulted from a structural mutation that affected channeling APS from the carboxyl-terminal sulfurylase activity to the amino-terminal kinase. (Lyle, S., et al., Sulfate-activating enzymes in normal and brachymorphic mice: evidence for a channeling defect, Biochemistry 34:940-45 [1995]).
A cDNA encoding a murine bifunctional ATP-sulfurylase/APS-kinase enzyme (known as Papss1, formerly known as Atpsk1) was isolated from brachymorphic mice and studied by mutational analysis. (Li, H. et al., The isolation and characterization of cDNA encoding the mouse bi-functional ATP sulfurylase-adenosine 5xe2x80x2-phosphosulfate kinase, J. Biol. Chem. 270(49):29453-59 [1995]; Deyrup, A. T. et al., Deletion and site-directed mutagenesis of the ATP-binding motif (P-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5xe2x80x2-phosphosulfate kinase enzyme, J. Biol. Chem. 273(16):9450-56 [1998]). Subsequently, bifunctional enzyes having ATP-sulfurylase (E.C. 2.7.7.4)/APS-kinase (E.C. 2.7.1.25) activities were designated xe2x80x9cPAPS synthetasexe2x80x9d (PAPSS; also known as ASAPK and ATPSK).
The bm mutation was placed on the mouse phenotypic map by Lane and Dickie (1968). Subsequent application of microsatellite markers to mice from a large backcross localized the bm mutation to a 2.5 cM region located at approximately 32 cM on mouse chromosome 19. (O""Brien, E. P., et al., Molecular map of chromosome 19 including three genes affecting bleeding time: ep, ru and bm, Mamm. Genome 5:356-60 [1994; Rusiniak, M. E., et al. (1996]). In addition, a liver cancer susceptibility gene, Hcs6, has been localized near bm in mouse strain C3H/He. (Manenti, G., et al. [1994]).
The bm mutation co-localized with a PAPSS gene. (Kurima, K. et al., A member of a family of sulfate-activating enzymes causes murine brachymorphism, Proc. Natl. Acad. Sci. 95(15):8681-85 [1998]; Sugahara, K. and Schwartz, N. B., Defect in 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate formation in brachymorphic mice, Proc. Nat. Acad. Sci. 76(12):6615-18 [1979]). Although brachymorphic mouse liver shows decreased PAPSS activity, the brachymorphic phenotype does not manifest any grossly recognizable liver defects. It has been observed that brachymorphic mouse liver has a decreased ability to esterify xenobiotic carcinogens with sulfate (Lyman, S. D. and Poland, A., Effect of the brachymorphic trait in mice on xenobiotic sulfate ester formation, Biochem. Pharmacol. 32:3345-50 [1983]), which implies that altered susceptibility to such agents could result from defects in a PAPSS gene. Consistent with this hypothesis, brachymorphic mice have decreased susceptibility to carcinogen-induced hepatocarcinoma, presumably due to a decreased ability to generate sulfated carcinogens. (Boberg, E. W., et al., Strong evidence from studies with brachymorphic mice and pentachlorophenol that 1xe2x80x2-sulfooxysafrole is the major ultimate electrophilic and carcinogenic metabolite of 1xe2x80x2-hydroxysafrole in mouse liver, Cancer Res. 43:5163-73 [1983]; Lai, C. C., et al., Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts, Carcinogenesis 8:471-78 [1987]).
PAPSS enzymes have been isolated and characterized in non-murine systems, for example, in association with rat chondrosarcomas. (Rosenthal, E. and Leustek, T., A multifunctional Urechis caupo protein, PAPS synthetase, has both ATP sulfurylase and APS kinase activities, Gene 165(2):243-48 [1995]; Schwartz, N. B., et al., Sulfate activation and transport in mammals; system components and mechanisms, Chem. Biol Interact. 109(1-3):143-51 [1998]; Lyle, S. et al., Rat chondrosarcoma ATP sulfurylase and adenosine 5xe2x80x2-phosphosulfate kinase reside on a single bifunctional protein, Biochemistry 33(19):5920-25 [1994]; Lyle, S. et al., Intermediate channeling between ATP sulfurylase and adenosine 5xe2x80x2-phosphosulfate kinase from rat chondrosarcoma, Biochemistry 33(22):6822-27 [1994]). Human cDNAs for PAPSS have also been cloned, and their activities have been analyzed biochemically and mutationally. (Yanagisawa, K. et al., cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5xe2x80x2-phosphosulfate kinase enzyme, Biosci. Biotechnol. Biochem. 62(5):1037-40 [1998]; Girard, J. P., et al., Sulfation in high endothelial venules: cloning and expression of the human PAPS synthetase, FASEB J. 12(7):603-12 [1998]; Venkatachalam, K. V. et al., Molecular cloning, expression, and characterization of human bifunctional 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate synthase and its functional domains, J. Biol. Chem. 273(30):19311-20 [1998]; Ventkatachalam, K. V., et al., Site-selected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate synthase, J. Biol. Chem. 274(5):2601-04 [1999]).
Phenotypically analogous to murine brachymorphism, is spondyloepimetaphyseal dysplasia (SEMD) in humans. SEMD is a subgroup of osteochondrodysplasias affecting skeletal development, linear bone and cartilage growth, and bone and cartilage maintenance. Effects of SEMD can include dwarfism, stunted or malformed limbs, enlarged joints, kyphoscoliosis (spinal warping), and brachydactyly (short fingers and toes). SEMD typically runs in families, and can be inherited in autosomal dominant or recessive manners. (Ahmad, M. et al., Distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred, Am. J. Med. Genet. 78(5):468-73 [1998]; Figuera, L. E., et al., Spondyloepimetaphyseal dysplasia (SEMD) Shohat type, Am. J. Med. Genet. 51(3):213-15 [1994]; Shohat, M. et al., New form of spondyloepimetaphyseal dysplasia (SEMD) in Jewish family of Iraqi origin, Am. J. Med. Genet. 46(4):358-62, [1993]; Whyte, M. P. et al., Hypotrichosis with spondyloepimetaphyseal dysplasia in three generations; a new autosomal dominant syndrome, Am. J. Med. Genet. 36(3):288-91 [1990]; Gertner, J. M., et al., Linkage studies of a Missouri kindred with autosomal dominant spondyloepimetaphyseal dysplasia (SEMD) indicate genetic heterogeneity, J. Bone Miner. Res. 12(8):1204-9, [1997]).
A nonsense mutation in a novel, cartilage-specific human PAPSS (PAPSS2, formerly ATPSK2), has been indicated as the cause of a recessive form of SEMD in an inbred Pakistani family. (Ahmad, M., et al., A distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred, Am. J. Med. Genet. 78:468-73 [1998]; ul Haque, M. F., et al., Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse, Nat. Genet. 20(2):157-62 [1998]). Genome-wide linkage studies localized the disease gene for this dwarfing condition to chromosome 10q23-24, in a region syntenic with the locus for the bm locus on mouse chromosome 19. (ul Haque, M. F., et al. [1998]). This disorder, designated SEMD Pakistani type, is characterized by short, bowed lower limbs, enlarged knee joints, kyphoscoliosis, a mild generalized brachydactyly, and early-onset degenerative joint disease in the hands and knees. Radiographs of patients with SEMD Pakistani type show delayed epiphyseal ossification, especially at the hips and knees, and platyspondyly.
Currently, there are only a few methods of detecting bone related diseases. (Eg., Klock, J. C., Chondroitin sulfate as a marker for bone resorption, U.S. Pat. No. 5,869,273; Takeshita, S. et al., Bone-related sulfatase-like protein and process for its production, U.S. Pat. No. 5,627,050). DNA-based diagnostic approaches have been suggested for some type 2 collagen disorders, such as Stickler syndrome, spondyloepiphyseal dysplasia, and achondrogenesis; achondroplasia (a defect in the fibroblast growth factor receptor 3 (FGFR3) gene); the collagen oligomeric matrix protein (COMP) disorders pseudoachondroplasia and multiple epiphyseal dysplasia, and others. (E.g., Ritvaniemi, Arthritis and Rheumatism 38:999-1004 [1995]; Shiang et al., Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia, Cell 78:335-42 [1994]; Briggs et al., Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage of oligomeric matrix protein gene, Nature Genetics 10:330-36 [1995]; Superti-Ferga et al., Recessively inherited multiple epiphyseal dysplasia with normal stature, club foot, and double layered patella caused by DTDST mutation, J. Med. Genet. 36:621-24 [1999]). But most reported diagnostic methods relate to osteogenic proteins. (E.g., Khandke, K. M., et al., Chromatographic process for the copurification of chondroitinase I and II proteins from Proteus vulgaris, U.S. Pat. No. 5,525,500; Parsons, T. F., et al., Osteogenic Factors, U.S. Pat. No. 5,106,626; Oppermann, H., et al., Cartilage and bone-inducing proteins, U.S. Pat. No. 5,750,651).
Accordingly, a reliable method is still needed for diagnosing SEMD, detecting the presence of SEMD in recessive carriers, and for treating osteoarthritic disorders, including osteochondrodysplasias, that are caused or aggravated by deficient PAPS synthetase activity. The present invention provides these and other advantages, as described herein.
The present invention relates to an isolated polynucleotide or to a nucleic acid construct that comprises a nucleic acid segment encoding a 3xe2x80x2-phosphoadenosine-5xe2x80x2-phosphosulfate (PAPS) synthetase (PAPSS), particularly, a human PAPSS2 nucleotide sequence of (SEQ. ID. NO.:1) or an orthologous murine Papss2 nucleotide sequence (SEQ. ID. NO.:2), sequences complementary to either one of them, degenerate coding sequences, or gene-specific fragments of (SEQ. ID. NOS.:1 and 2). The present polynucleotides and nucleic acid constructs containing PAPSS2 and Papss2 nucleotide sequences include RNA; DNA; and chimeric RNA/DNA. Embodiments include probes, primers, and expression vectors containing PAPSS2- and Papss2-specific nucleotide sequences.
The present invention also relates to a genetically modified vertebrate cell containing a nucleic acid construct of the present invention and to a non-human vertebrate containing the cell. The present invention also relates to human PAPSS2 and murine Papss2 proteins encoded by the present polynucleotide or nucleic acid construct, including fusion proteins that contain, together with a PAPSS2 or Papss2 amino acid sequence, any other predetermined polypeptide sequence. The present invention also relates to antibodies and antibody fragments that selectively bind the PAPSS2 or Papss2 protein.
The present invention is also directed to a method of diagnosing spondyloepimetaphyseal dysplasia (SEMD) in a human subject. The method involves amplifying nucleic acids from a sample that define an PAPSS2 gene sequence, or a gene-specific fragment thereof; and analyzing the amplified nucleic acids for the presence of homozygosity for a variant allele of PAPSS2. The sample is a bodily substance containing human nucleic acid, for example a blood sample, obtained from a human subject having at least one symptom of SEMD. Homozygosity for a variant allele of PAPSS2 is diagnostic for SEMD in the human subject. In particular, the present invention also relates to a method of diagnosing SEMD Pakistani-type in a human subject. But the present invention also relates to a method of identifying a heterozygous human carrier of an SEMD-associated allele.
The present invention provides a genetic testing kit for diagnosing SEMD or for identifying a human carrier of SEMD, including SEMD Pakistani-type. The genetic testing kit contains oligonucleotide primers of the present invention.
Also, nucleic acid constructs of the present invention are used in a method of gene therapy for treating a human subject having an osteoarthritic disorder that is caused or aggravated by deficient enzymatic sulfation activity. The present invention is also related to a protein therapy method for treating a human subject having an osteoarthritic disorder that is caused or aggravated by deficient enzymatic sulfation activity. This method employs the inventive PAPSS2 fusion protein.